Bachmeier Beatrice E, Ruoss Isabell, Hagedorn Hjalmar G, Nerlich Andreas G
Department of Clinical Chemistry and Clinical Biochemistry, Ludwig-Maximilians-University, Munich, Germany.
Int J Mol Med. 2002 Oct;10(4):371-6.
We investigated three clonally related human keratinocyte cell lines of different biological behaviour, HaCaT (non-tumorigenic), A5 (benign, tumorigenic) and II-4RT (malignant, tumorigenic), with regard to the expression of TGF-beta-isoforms -1, -2 and -3 and that of the TGF-beta-cell-receptors TBR-I, -II and -III. In addition, we amplified and sequenced the genome of TBR-II which is known to be a target for mutations in several types of malignant tumors including squamous cell carcinomas. In all three cell lines, TGF-beta1 and -beta3 were present only in very low amounts. Western blots provided no evidence for differences in TGF-beta1 between the cell lines. However, in immunohistochemistry more cells were slightly positive for this cytokine in HaCaT than in A5 and II-4RT cells. In contrast, a significantly variable expression of TGF-beta2 was seen by both Western blot and immunohistochemistry. Thereby, the non-tumorigenic HaCaT-cells contained significantly more TGF-beta2 than the tumorigenic, benign A5 cells and the malignant II-4RT cells. TBR-I, -II and -III were present in all three cell lines. While most cells were positive for TBR-I, only part of the cells contained TBR-II and -III, however, without obvious differences between the three cell lines. The molecular analysis of all 7 exons of TBR-II by PCR amplification and direct sequencing revealed in all three cell lines correct sequences without evidence for mutations. Our study indicates differences in the expression of TGF-beta in a human model of keratinocytes of varying tumorigenicity, but presents no evidence for mutations in the functionally most important TGF-beta-receptor TBR-II. This suggests a dysregulation of cytokine control on the level of TGF-beta expression, which may be responsible for the biological behaviour.
我们研究了三种具有不同生物学行为的克隆相关人类角质形成细胞系,即HaCaT(非致瘤性)、A5(良性,致瘤性)和II - 4RT(恶性,致瘤性),检测了转化生长因子β(TGF-β)异构体-1、-2和-3以及TGF-β细胞受体TBR-I、-II和-III的表达情况。此外,我们扩增并测序了TBR-II的基因组,已知它是包括鳞状细胞癌在内的几种恶性肿瘤中发生突变的靶点。在所有这三种细胞系中,TGF-β1和-β3的含量都非常低。蛋白质免疫印迹法未发现细胞系之间TGF-β1存在差异。然而,免疫组织化学显示,与A5和II - 4RT细胞相比,HaCaT细胞中对这种细胞因子呈弱阳性的细胞更多。相比之下,蛋白质免疫印迹法和免疫组织化学均显示TGF-β2的表达存在显著差异。由此可见,非致瘤性的HaCaT细胞所含的TGF-β2明显多于致瘤性的良性A5细胞和恶性II - 4RT细胞。所有三种细胞系中均存在TBR-I、-II和-III。虽然大多数细胞对TBR-I呈阳性,但只有部分细胞含有TBR-II和-III,不过这三种细胞系之间没有明显差异。通过聚合酶链反应(PCR)扩增和直接测序对TBR-II的所有7个外显子进行分子分析,结果显示所有三种细胞系的序列均正确,未发现突变证据。我们的研究表明,在具有不同致瘤性的人类角质形成细胞模型中,TGF-β的表达存在差异,但未发现功能上最重要的TGF-β受体TBR-II发生突变的证据。这表明细胞因子控制在TGF-β表达水平上存在失调,这可能与生物学行为有关。